A major focus of the Waldmann Laboratory is the definition of the molecular abnormalities of T-cell malignancy. In collaboration with Mark Raffeld and Elaine Jaffe, Hematopathology Laboratory, frequent STAT5B mutations were demonstrated in the malignant cells of hepatosplenic T-cell lymphomas. RNA sequencing on purified leukemic cells of 27 patients with ATL and 11 ATL cell lines from patients' paired cells that remain in the germline configuration we demonstrated a number of molecular abnormalities. Five patient ATL cells/lines manifested NOTCH1 mutations. There were six CARD11 missense mutations. Furthermore there were four nonsense mutations of CCR4 observed in 8 ATL samples (18%). CCR4 is a chemokine receptor highly expressed in ATL cells and in normal TH17 and T reg cells. It is the target of our new clinical trial in patients with ATL using the KW-0761 monoclonal antibody. Nine recurrent somatic mutations of CCR4 encoding CCR were detected in 14/53 ATL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C299, Q330 and Y331 in the carboxyl terminal. Functionally the CCR4-Q330, a nonsense isoform, with gain of function since it increased cell migration towards the CC4R ligands, CCL17 and CCL22 in part by impairing receptor internalization. This mutant enhanced PI (3) kinase/AKT activation following receptor engagement by CCL22 in ATL cells and conferred a relative advantage in long-term in vitro cultures. These findings implicate somatic gain of function CCR mutations in the pathogenesis of ATL. A dominant element in our efforts to define the cellular abnormalities in ATL were focused on the disordered yc cytokine receptor (IL-2, IL-2R, IL-15, IL-15R and IL-9) signaling (JAK1, JAK3, STAT5) in ATL. Previously we demonstrated that HTLV-1 Tax activation in T cells in vivo and in vitro is associated with constitutive IL-2 and IL-2R alpha expression, IL-15, IL-15R alpha expression and a paracrine stimulatory loop that involves IL-9 production by ATL cells. To extend these observations we performed molecular interference Achilles' heel screening of 14 ATL cell lines employing a library of retroviral vectors for inducible expression of short hairpin RNAs (shRNAs) to identify genes essential for leukemic cell survival. Using this process of loss-of-function screen, 6 of 7 distinct cytokine dependent ATL cell lines were shown to be critically dependent on JAK1 and JK3 for proliferation and survival. The critical nature of the IL-2, IL-9, IL-15, JAK1 and JAK3 pathway for ATL was supported by our observation that the ex vivo 6-day culture of PBMCs from patients with smoldering and chronic ATL could be inhibited by the administration of antibodies to IL-2 receptor, IL-15 and IL-9. Furthermore, we demonstrated that the pan JAK inhibitor, tofacitinib and the JAK1/2 inhibitor, ruxolitinib inhibited the ex vivo proliferation of PBMCs from ATL patients by over 60%. On the basis of this study showing the critical nature of the gamma cytokine JAK/STAT system in ATL we have initiated a clinical trial involving the JAK1/2 inhibitor, ruxolitinib in patients with ATL. Furthermore, we are searching for novel multicomponent/combination drug therapies for ATL by applying high-throughput matrix screening for cellular signaling on ATL cell lines to define combination therapies that identify agents with synergy. Optimal synergy was demonstrated between the JAK1/2 inhibitor, ruxolitinib added in association with the Bcl-xL inhibitor, navitoclax. Examination of the mechanistic underpinnings of this combination highlighted a stimulation Bim and Puma expression and reduced phosphorylation of Bad upon cellular exposure to ruxolitinib. The combination was further noted to strongly activate BAX effect mitochondrial depolarization that led to PARP and Mcl-1 cleavage. Ruxolitinib and navitoclax independently demonstrated modest antitumor efficacy, while the combination dramatically lowered tumor burden and prolonged survival in an aggressive ATL murine model. Critically this combination strongly blocked proliferation of ex vivo cultures of 5 ATL patients' PBMCs. These studies provide support for a therapeutic trial in patients with smoldering and chronic ATL using a combination of agents that inhibit JAK1/3 and the antiapoptotic Bcl-2 family members.